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Gewählte Publikation:

Lingg, N; Cserjan-Puschmann, M; Fischer, A; Engele, P; Kross, C; Schneider, R; Brocard, C; Berkemeyer, M; Striedner, G; Jungbauer, A.
(2021): Advanced purification platform using circularly permuted caspase-2 for affinity fusion-tag removal to produce native fibroblast growth factor 2
J CHEM TECHNOL BIOT. 2021; 96(6): 1515-1522. FullText FullText_BOKU

Abstract:
BACKGROUND Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N-terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion-tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N-terminus. RESULTS Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed-batch cultivations. The protein of interest (POI) carried an N-terminal affinity fusion-tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion-tag with a circularly permuted human caspase-2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N-terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 mu g mL(-1) dsDNA and similar to 1000 EU mL(-1) endotoxin. Only a small number of E. coli host cell proteins were co-purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off-target cleavage could be observed. CONCLUSION Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion-protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2-based platform fusion protein process (CASPON technology) purification platform. (c) 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Autor*innen der BOKU Wien:
Cserjan Monika
Jungbauer Alois
Lingg Nico
Striedner Gerald

Find related publications in this database (Keywords)
Bioseparations
downstream
proteins
chromatography
enzymes


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